Fine tuning of glutamatergic synaptic transmission underlies information processing in the brain. Exquisite regulatory control arises from subsynaptic protein organization at the nanometer scale. Though key aspects of molecular organization within the active zone and postsynaptic density are well known, our lab recently reported a novel coordination of nanoscale organization between pre- and postsynaptic specializations at the synapse. Clusters of proteins establishing presynaptic sites of action potential-evoked neurotransmitter release (particularly RIM1/2) are aligned across the synapse with postsynaptic nanoclusters of glutamate receptors and scaffold proteins such as PSD-95. This alignment is spatially confined to ~80 nm in diameter, much smaller than the size of the entire synapse. This `nanocolumn' organization is expected to increase synaptic strength by enhancing the probability of receptor activation during synaptic transmission (Tang et al., 2016), and may reveal a modular structure important for synaptic plasticity. Thus, understanding the biology governing this trans- synaptic alignment could prove useful for our understanding of diseases and disorders in which synaptic transmission is known to be disrupted, including schizophrenia and autism spectrum disorders. Nevertheless, the mechanism by which this trans-cellular alignment is established is unknown. One unexplored explanation with important implications for synaptic function involves the glutamate receptors themselves. AMPA receptors and NMDA receptors are arranged in nanoclusters and are enriched within the nanocolumn. Furthermore, they interact with PSD-95 intracellularly and their large extracellular domains are known to bind numerous trans- synaptic proteins found in the synaptic cleft. Given these observations, I hypothesize that glutamate receptors mediate trans-synaptic alignment of RIM1/2 and PSD-95 nanoclusters. To test this, I first will use established mouse lines as well as knockdown experiments in culture to examine the protein distribution of synapses entirely lacking in ionotropic glutamate receptors. I will conduct three dimensional direct stochastic optical reconstruction microscopy (3D dSTORM) and expansion microscopy to assess trans-synaptic alignment, and whole-cell patch clamp electrophysiology to validate the loss of AMPA and NMDA currents. Should alignment be disturbed, I will then explore which glutamate receptors are responsible for alignment. Second, I will test whether receptor position within the synapse is sufficient to dictate presynaptic protein organization. To do this, I will use a published optical dimerization technique to recruit glutamate receptors to both central and peripheral sites within the postsynaptic density. Following recruitment, I will use 3D dSTORM to determine if alignment of RIM1/2 nanoclusters is dependent on positioning of recruited receptors. Glutamate receptors, though obviously critical for synaptic transmission, are often thought to be simply passive contributors to synapse function as they sit within the synapse or diffuse in and out. My experiments will test a dramatically different view of the receptors: that they actively shape the structure of synapses and thus likely control their own activation.